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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:519-524.)
© 1999 American Heart Association, Inc.

Original Contributions

Atherosclerotic Aortic Gangliosides Enhance Integrin-Mediated Platelet Adhesion to Collagen

Fei-Qiu Wen; Adnan A. Jabbar; Dharmesh A. Patel; Tamara Kazarian; Leonard A. Valentino

From the Department of Pediatrics, Rush Medical College and Rush Children's Hospital, Chicago, Ill.

Correspondence to Leonard A. Valentino, MD, Rush Children's Hospital, 1653 W Congress Parkway, Chicago, IL 60612-3833. E-mail lvalentino{at}rush.edu

	Abstract

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Abstract—Gangliosides, sialic acid–containing glycosphingolipids, accumulate in atherosclerotic vessels. Their role in the pathogenesis of atherosclerosis is unknown. Gangliosides isolated from tumor cells promote collagen-stimulated platelet aggregation and ATP secretion and enhance platelet adhesion to immobilized collagen. These activities are all mediated by ganglioside effects on the platelet integrin collagen receptor ₂ß₁. Therefore, we hypothesized that gangliosides isolated from atherosclerotic plaques would enhance platelet adhesion to immobilized collagen, a major component of the subendothelial matrix of blood vessels. Furthermore, we questioned whether this effect of atherosclerotic gangliosides might play a role in the pathogenesis of atherosclerosis. To test this hypothesis, we isolated the gangliosides from postmortem aortas of patients with extensive atherosclerotic disease and examined their effects on platelet adhesion. Samples of aortic tissue taken from areas involved with atherosclerotic plaque demonstrated accumulation of gangliosides (64.9±6.5 nmol/g wet weight) compared with gangliosides isolated from control normal aortic tissue taken from children who died of noncardiac causes (NAGs; 21.1±6.4 nmol/g wet weight). Interestingly, samples of tissue taken from diseased aortas but from areas not involved with gross plaque formation also demonstrated ganglioside accumulation (47.6±12.8 nmol/g wet weight). Next, the activity of each of these gangliosides on platelet adhesion to immobilized type I collagen was studied. Atherosclerotic aortic gangliosides (AAGs) as well as those isolated from grossly unaffected areas of the same aorta (UAGs) both increased platelet adhesion compared with control NAGs (OD₅₇₀, 0.37±0.11 and 0.29±0.14 versus 0.16±0.07, respectively; P<0.01 and P<0.05, respectively). These OD₅₇₀ values corresponded to 9x10⁵, 8x10⁴, and 6x10³ platelets per well after preincubation with 5 µmol/L AAG, UAG, and NAG, respectively. Increased adhesion was observed after preincubation with as little as 0.5 µmol/L AAG, and maximal adhesion was seen at 2.5 µmol/L, with a plateau extending to the highest concentration tested, 10 µmol/L. The effect of AAGs on platelet adhesion to collagen was abrogated by incubation of treated platelets with F-17 anti-₂ monoclonal antibody (OD₅₇₀, 0.13±0.02). Finally, the effects of the major individual gangliosides isolated from atherosclerotic tissues, G_M3 and G_D3, were tested. G_M3 increased adhesion to collagen (OD₅₇₀, 0.415±0.06) as did G_D3 (0.31±0.08). Similar to that of AAGs, the effect of both molecules was blocked by F-17 (0.09±0.04 and 0.13±0.06, respectively). These experiments demonstrate that accumulated atherosclerotic gangliosides promote platelet adhesion to collagen, the major component of the subendothelial matrix. Furthermore, this activity is mediated by an effect of the gangliosides on the collagen-binding integrin ₂ß₁. This activity may provide a mechanism for the development of platelet thrombi at sites where atherosclerotic gangliosides accumulate and help to explain the role of platelets in the process of atherosclerotic disease progression.

Key Words: atherosclerosis • gangliosides • platelets • integrins

	Introduction

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Platelets and platelet-rich thrombi play a major role in atherosclerosis.^{1 2 3} Chemical or mechanical injury to endothelial cells of the vascular system stimulates platelet–vessel wall interaction. As a result, platelets, and subsequently fibrin, become part of the atherosclerotic plaque, leading to progression of the lesion. Gangliosides, sialic acid–containing glycosphingolipids, accumulate in atherosclerotic vessels.⁴ The chromatographic profile of the gangliosides isolated from the atherosclerotic plaques is markedly different compared with those from the unaffected intima.⁵ For example, 93% of the gangliosides isolated from intimal plaque are G_M3 versus 66% in the unaffected intima. The content of G_D3 ganglioside in the underlying media of atherosclerotic lesions is also increased more than 3-fold compared with unaffected aortic media.⁵ In contrast, G_D3 comprises 2.6% of the intimal plaque gangliosides compared with 22% in normal intima.⁵

Gangliosides play a role in cell-cell interaction, as well as in cell growth and proliferation,⁶ all processes important in the pathogenesis of atherosclerosis. Gangliosides are synthesized in the Golgi and transported to the cell membrane, where they may interact with other membrane proteins or be shed from the surfaces of cells and enter the circulation.^{7 8} Circulating gangliosides are transported in association with lipoproteins, primarily LDL.⁹ Atherosclerotic gangliosides modify the surface structure and stimulate the aggregation of LDL particles, and ganglioside-modified LDL is easily recognized and taken up by macrophages.¹⁰ Oxidative modification of LDLs and their non–receptor-regulated uptake by monocytes/macrophages may play a key role in the formation of fatty streaks and early atheromatous lesions.¹¹ Gangliosides also affect platelet function. For example, gangliosides isolated from neuroblastoma tumor cells enhance platelet aggregation and activation¹² as well as promote platelet adhesion to extracellular matrix collagen.¹³ These effects are mediated through the integrin collagen receptor ₂ß₁. Collagen is a principle component of atherosclerotic plaque¹⁴ and in the vessel wall is thought to be the major substrate for platelet adhesion. The role of atherosclerotic gangliosides in the interaction of platelets with collagen has not been evaluated. The objectives of this study were to determine whether atherosclerotic gangliosides enhance platelet adhesion to collagen and, if so, whether the effect is due to ganglioside modulation of platelet ₂ß₁ integrin function.

	Methods

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Materials
Type I rat tail collagen from Sigma Chemical Co was used as the substrate for platelet adhesion experiments. Anti-₂ monoclonal antibody F-17 was a gift from Dr Harvey Gralnick (Hematology Service, National Institutes of Health). The bicinchoninic acid (BCA) protein assay reagent was obtained from Pierce, and other chemicals were from Fisher.

Preparation of Human Aortic Tissue
Samples of aorta were taken aseptically 2 to 7 hours postmortem from 5 patients, 58 to 77 years old, of whom 3 were male. All patients had extensive (types IV and V) atherosclerosis involving the aorta in addition to other vessels. The aorta was cut longitudinally to identify the type of atherosclerotic lesion. Grossly uninvolved portions of aorta and areas with fatty streaks and atherosclerotic plaques were excised, placed on ice, and washed to remove blood clots. Control tissue was obtained from the same region of aortas from 4 children (newborn to 16 years of age) who had died of noncardiac, nonmetabolic causes and at autopsy were without gross or microscopic evidence of atherosclerosis. This project was approved by the Institutional Review Board of Rush University.

Extraction and Purification of Gangliosides
Tissues were minced, homogenized at 4°C, and then lyophilized. The total gangliosides were isolated as previously described.¹⁵ In brief, the total lipids were extracted twice with 20 volumes of chloroform/methanol (1:1), and the extracts were then combined, dried by rotoevaporation, redissolved in a small volume of chloroform/methanol (1:1), and stored overnight at -20°C. Insoluble glycoproteins were removed by centrifugation (1000g, 4°C), and the supernatant was dried under a stream of N₂. The gangliosides were isolated by partitioning the dried total lipid extract in diisopropyl ether/1-butanol/water (6:4:5, vol/vol/vol),¹⁶ and traces of salts and other low-molecular-weight contaminants were removed by Sephadex G-50 gel exclusion chromatography. The gangliosides were further purified by normal-phase high-pressure liquid chromatography on a Hibar RT LiChrosorb silicon NH₂ column.¹⁵ The total and individual ganglioside fractions were collected, lyophilized, and repurified by Sephadex G-50 gel exclusion chromatography. Gangliosides were quantified as nanomoles of lipid-bound sialic acid (LBSA) by a modification (Miettinen et al¹⁸ ) of the method of Svennerholm¹⁷ and visualized by high-performance thin-layer chromatography as purple bands by using resorcinol reagent.¹⁹

Platelet Isolation
Platelet donors abstained from all medications for a minimum of 7 days, fasted overnight, and provided written, informed consent. Platelets were isolated from donors according to the methods of Mustard et al.²⁰ In brief, blood was drawn into tubes (Becton Dickinson) containing acid-citrate-dextrose–A (1:6, vol/vol) to which 35 U/mL preservative-free heparin was added. Platelet-rich plasma was isolated by centrifugation (3000g, 15 minutes at 22°C) and then passed over Sepharose 2B (Pharmacia) in modified Tyrode's buffer (MTB) in the absence of Ca²⁺ or Mg²⁺ according to the methods of Coller et al.²¹

Platelet Adhesion Assay
Platelet adhesion was determined by the methods of Ill et al²² and Santoro²³ as modified by Coller et al.²¹ Collagen fibers were diluted with isotonic glucose (pH 2.7 to 2.9) to a concentration of 40 µg/mL. One hundred microliters of this suspension was used to coat the wells of a polystyrene microtiter plate (Falcon 3915, Becton Dickinson) overnight at 22°C. The wells were aspirated and blocked with 100 µL of 0.5% BSA solution for 1 hour at 22°C and then washed 3 times with MTB. Control wells were coated with BSA alone. Gel-filtered platelets were adjusted to 10⁵/µL in MTB without MgC1₂ and incubated with purified aortic gangliosides at the specified concentration for 30 minutes at 37°C with gentle mixing. The platelets were washed once to remove unbound gangliosides and resuspended in MTB with 2.56 mmol/L MgC1₂, and 100 µL of the final platelet suspension was added to the wells and incubated for 1 hour at 37°C with gentle mixing. The wells were vigorously washed 5 times with 100 µL MTB to remove nonadherent platelets and loose aggregates. The number of adherent platelets was determined by using the BCA protein assay as described by Tuszynski and Murphy.²⁴ In brief, adherent platelets were solubilized with 100 µL BCA protein assay reagent and incubated for 1 hour at 37°C. Absorbance was measured at 570 nm (OD₅₇₀) with a microtiter plate reader (Biotech). In each experiment, a standard curve of OD₅₇₀ and platelet number was constructed by adding platelets (10³ to 10⁶ per well) to collagen-coated, BSA-blocked wells as described above. An average OD₅₇₀ value was calculated from triplicate wells over the range of platelet concentrations and related to direct phase-contrast microscopy counts by linear regression analysis. Nonspecific platelet adhesion to BSA typically resulted in OD₅₇₀ values <0.05. In all experiments, a direct correlation was observed between measured OD₅₇₀ and platelet number (r²>0.95). A representative standard curve (r²=0.9798) from a typical experiment is shown in the insert of Figure 3. OD₅₇₀ values of 0.1, 0.2, 0.3, and 0.4 represent platelet numbers of 1.76x10³, 1.57x10⁴, 1.39x10⁵, and 1.26x10⁶, respectively.

View larger version (12K): [in this window] [in a new window]   Figure 3. Effect of human aortic gangliosides on platelet adhesion to collagen. Gel-filtered platelets were preincubated with 5 µmol/L AAGs, UAGs, or NAGs for 30 minutes at 37°C with gentle mixing. Unbound gangliosides were removed by centrifugation, and the final platelet product was resuspended to 105/µL in MTB with 2.56 mmol/L MgCl2. One hundred microliters of the platelet suspension was added to collagen-coated (4 µg per well), BSA-blocked wells and allowed to incubate for 1 hour 37°C with gentle mixing. After removal of nonadherent platelets, the residual adherent platelet number was estimated by the BCA assay. The mean and SD of 4 experiments performed in 6 replicates are shown. Insert, Standard curve of platelet number. Collagen (4 µg per well) was coated onto wells of a 96-well non–tissue culture–treated plate and then blocked with BSA as described in Methods. Aliquots of l03 to l06 gel-filtered platelets (enumerated by direct microscopic examination) were added to triplicate wells, and protein content was determined by the BCA assay as described in Methods. In the standard wells, all added platelets were present in the wells when the BCA assay was performed. Color intensity was read on a Biotech ELISA plate reader with a 570-nm filter. The mean and SD of triplicate values from a single experiment are shown. In this example, r2 was 0.9798 by linear regression analysis. This experiment is representative of >70 individual experiments performed with gel-filtered platelets.

	Results

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Ganglioside Content and Composition in Aortic Tissues
Figure 1 demonstrates a substantial increase in the total ganglioside content of atherosclerotic aortic compared with control normal aortic tissue. The ganglioside content of aortic tissue with gross plaque was 64.9±6.5 nmol/g tissue wet weight (n=5) and is similar to adjacent tissue that was grossly uninvolved (47.6±12.8, n=4) but is 3 times greater than that of control normal aorta (21.1±6.4, n=4). The difference in ganglioside content between aortic tissue with gross plaque and adjacent tissue that was grossly uninvolved was not significant, but both were significantly greater than control normal aorta (P<0.001). The chromatographic profile of these gangliosides is shown in Figure 2. Lanes were loaded with the gangliosides isolated from identical amounts of tissue (60 mg wet weight), resulting in 3.12, 1.66, and 1.16 nmol LBSA in lanes 1, 2, and 3, respectively. Densitometric analysis demonstrated excess G_M3 (268%) and G_D3 (143%) visible in atherosclerotic aortic tissue (lane 1) compared with control normal tissue (the G_M3 and G_D3 bands in lane 3 were normalized to 100% comparison). Interestingly, although there was an increase of G_M3 in the grossly uninvolved aortic tissue (lane 2), there was no difference in G_D3 compared with control normal tissue, suggesting that biochemical changes leading to accumulation of G_M3 precede those leading to accumulation of G_D3 ganglioside. Atherosclerotic G_M3 migrated as a doublet. A prominent lower band of G_M3 was present in atherosclerotic aortic tissue (lane 1) and was also clearly visible in tissue from uninvolved areas of the same aorta (lane 2) but was essentially absent from control normal tissue (lane 3).

View larger version (12K): [in this window] [in a new window]   Figure 1. Ganglioside content of human aortic tissues. Column AAG depicts the ganglioside content of aortic tissues from 5 patient samples with gross atherosclerotic plaque. Column UAG depicts the ganglioside content of 4 patient samples taken from grossly uninvolved areas adjacent to atherosclerotic plaque. Column NAG depicts the ganglioside content of aortic tissue from the 4 children without gross or microscopic evidence of atherosclerotic disease. Ganglioside content is given in nmol LBSA per g tissue wet weight.

View larger version (50K): [in this window] [in a new window]   Figure 2. Chromatographic profile of gangliosides isolated from human aortic tissue. Lane S, standard gangliosides; lane 1, AAGs; lane 2, UAGs; and lane 3, NAGs.

Effect of Aortic Gangliosides on Platelet Adhesion to Collagen
Figure 3 demonstrates that preincubation of platelets with 5 µmol/L gangliosides isolated from both atherosclerotic aortic plaque (AAG) and adjacent uninvolved aortic tissue (UAG) increased platelet adhesion to collagen compared with platelets preincubated with MTB buffer (OD₅₇₀ 0.37±0.11 and 0.29±0.14 versus 0.13±0.03, respectively; P<0.01 and P<0.05, respectively). Gangliosides isolated from the normal aorta (NAGs) did not increase platelet adhesion (OD₅₇₀ 0.16±0.07) compared with MTB control (P>0.05).

Figure 4 demonstrates the effect of AAG concentration on platelet adhesion. A significant increase in adhesion was observed after incubation of platelets with even as little as 0.5 µmol/L AAG compared with platelets incubated with MTB buffer (OD₅₇₀ 0.21±0.02 versus 0.12±0.05, P<0.01). Maximal adhesion was observed at a concentration of 2.5 µmol/L AAG (OD₅₇₀ 0.38±0.07) and was sustained up to 10 µmol/L, the highest concentration tested. The effects of UAG and NAG on platelet adhesion are also shown. Preincubation of platelets with 2.5 µmol/L UAG increased platelet adhesion compared with NAG (OD₅₇₀ 0.26±0.01 versus 0.17±0.01, P<0.01), but this increase was less than with AAG. Increasing the concentration of UAG resulted in increased platelet adhesion. At 10 µmol/L, there was no difference in the adhesion of platelets exposed to AAG compared with UAG. In contrast, increasing the concentration of NAG did not result in more platelet adhesion.

View larger version (13K): [in this window] [in a new window]   Figure 4. Effect of ganglioside concentration on platelet adhesion. Gel-filtered platelets were preincubated with 0.5 to 10 µmol/L AAGs, UAGs, NAGs, or MTB in the absence of MgCl2 for 30 minutes at 37°C. The remainder of the experiment was performed as in the legend to Figure 3. The mean and SD of 3 experiments performed in 6 replicates are shown.

AAGs Interact With Platelets by an ₂ß₁-Dependent Mechanism
₂ß₁ Integrin is the major collagen receptor on platelets. Our prior experiments indicated that gangliosides from neuroblastoma tumor cells promoted platelet adhesion to collagen by an ₂ß₁-integrin–dependent effect.¹³ To determine whether a similar mechanism was operative with atherosclerotic gangliosides, adhesion experiments were performed with anti-₂ monoclonal antibody to block the ₂ß₁ receptor. In Figure 5, adhesion of AAG-preincubated platelets was reduced to control levels by F-17 anti-₂ antibody (OD₅₇₀ 0.13±0.02 versus 0.14±0.03, P>0.05).

View larger version (12K): [in this window] [in a new window]   Figure 5. Effect of anti-2 antibody on AAG-enhanced platelet adhesion to collagen. Gel-filtered platelets were preincubated for 30 minutes with 1 µmol/L AAGs. Unbound gangliosides were removed by washing, and the final platelet product was resuspended to 105/µL in MTB containing 10 µg/mL F-17 anti-2 antibody and 2.56 mmol/L MgCl2 and then incubated for 30 minutes at 37°C. Excess antibody was not washed away. One hundred microliters of platelets was added to collagen-coated, BSA-blocked wells and incubated for an additional 60 minutes at 37°C with gentle mixing. Nonadherent platelets were removed by washing, and the residual adherent platelet number was estimated by the BCA assay. The mean and SD from 3 experiments performed in 6 replicates are shown.

Effect of Atherosclerotic G_M3 and G_D3 Gangliosides on Platelet Adhesion to Collagen
As Figure 2 demonstrated, the major individual gangliosides in atherosclerotic aortic tissue are G_M3 and G_D3, which together compose >80% of total gangliosides in fatty streaks and atherosclerotic plaques.⁴ To determine whether G_M3 and G_D3 play a role in ₂ß₁-integrin–mediated platelet adhesion to collagen, the effects of these gangliosides were studied. G_M3 isolated from AAGs increased platelet adhesion to a greater extent compared with G_M3 isolated from NAG (OD₅₇₀ 0.44±0.09 versus 0.28±0.05, P<0.001; Figure 6, solid bars). G_M3 from either source increased adhesion compared with MTB control (0.13±0.03, open bar). The same was true for G_D3 isolated from AAG and NAG (0.31±0.08 and 0.25±0.06, respectively, P<0.05; Figure 6, hatched bars). The enhancing effect of G_M3 and G_D3 (isolated from AAGs) was abrogated by anti-₂ antibody (OD₅₇₀ 0.09±0.04 and 0.13±0.06, respectively; P>0.05 and P>0.05, respectively).

View larger version (16K): [in this window] [in a new window]   Figure 6. Effect of atherosclerotic GM3 and GD3 on platelet adhesion to collagen. Gel-filtered platelets were preincubated for 30 minutes with 1 µmol/L GM3 or GD3 isolated from AAGs (solid bars) or NAGs (hatched bars). The effect of anti-2 blocking antibody on GM3- and GD3-enhanced platelet adhesion was examined as described in the legend to Figure 5. Buffer control is shown in the open bar. The mean and SD from 5 experiments performed in 6 replicates are shown.

	Discussion

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Our study demonstrates that there are significant differences in the ganglioside content and composition of human atherosclerotic aorta. In addition to a 3-fold increase in ganglioside content of the atherosclerotic aorta, the ganglioside composition is also altered, with a prominent increase in G_M3 and, to a lesser degree, G_D3. The ganglioside content of grossly uninvolved aortic tissue from atherosclerotic patients was increased >2-fold compared with control normal aortic tissue without atherosclerosis (47.6±12.8 versus 21.05±6.4 nmol/g tissue wet weight, P<0.001). Attempts to identify age- and sex-matched control tissue were unsuccessful. Only when aortas of young children were examined could we identify tissues lacking the early stages of atherosclerosis. This observation is consistent with those of Berenson et al,²⁵ who found that the majority of individuals <40 years of age who die of noncardiac causes have fatty streaks and fibrous plaques in their coronary arteries and aortas. Therefore, tissues from young children were used to isolate control normal "nonatherosclerotic" gangliosides. Our results are consistent with those of Hara and Taketomi,²⁶ who found that the aortic ganglioside content in Watanabe heritable hyperlipidemic (WHHL) rabbits was markedly elevated, 12-fold greater, than in normal rabbits. Mukhin et al⁴ and Prokazova et al⁵ obtained similar results by examining human aortic tissue. Similar to our data, G_M3 contributed most to the increase in atherosclerotic gangliosides. G_D3, which is almost undetectable in normal aortic tissue, was also increased in the WHHL rabbit aorta²⁶ and in the human tissue examined (References 4 and 5^{4 5} and our data). Combined, these results suggest that there are biochemical changes resulting in ganglioside accumulation before plaque formation. Samples of aortic tissue taken from areas that grossly appeared not to be involved with atherosclerotic plaque but that were adjacent to a plaque had 47.6±12.8 nmol LBSA/g wet weight tissue compared with 64.9±6.5 nmol LBSA/g wet weight tissue, a difference of only 27%. Therefore, despite the lack of evidence for atherosclerotic involvement, clear biochemical abnormalities exist, supporting the notion that ganglioside accumulation precedes the anatomic and histological changes characteristic of atherosclerosis. Biochemical alterations result in accumulation of gangliosides with aberrant lipid and carbohydrate structures. These enzymatic differences are not well characterized.

Vascular injury and thrombus formation are key events in the origination and progression of atherosclerosis. Vascular injury is classified into 3 pathological types.²⁷ Type I injury consists of functional alterations in endothelial cells without substantial morphological changes. Blood flow shear stress causes chronic injury to the endothelium, especially at bending sites or branching points.²⁸ Accumulation of lipids and infiltration of monocytes (macrophages) in the damaged areas are characteristic. Factors such as hypercholesterolemia, oxidized LDL, inflammatory mediators, oxygen-derived free radicals, and vasoactive amines are considered to potentiate the endothelial injury. Type II injury is defined by endothelial denudation and intimal damage, but with an intact internal elastic lamina. Platelet adhesion to exposed subendothelial collagen is a prominent feature of this type of lesion. Platelets, together with macrophages and endothelial cells, may release the various factors described above. These lead to the simultaneous migration and proliferation of smooth muscle cells, a process that may contribute to the formation of either a "fibrointimal lesion" or the outer capsule of a "lipid lesion."²⁹ Type III injury is characterized by damage through the intima into the media, with thrombus formation.²⁷ When thrombi are small, they become organized and contribute to the rapid growth of the atherosclerotic plaque.^{29 30} Disruption or erosion of a plaque leads to thrombosis and acute vascular compromise. Therefore, it is clear that platelets and platelet-rich thrombi are major contributors to the development, propagation, and clinical presentation of atherosclerosis.

Gangliosides are found in all eukaryotic cell membranes. Structurally, these molecules have a water-soluble carbohydrate head group and a lipophilic tail. There is potential for enormous structural diversity in each portion of the molecule. Differences in the type of oligosaccharide moieties as well as the number, position, and linkage of sialic acid residues, combined with variations in ceramide structure and fatty acyl hydroxylation, lead to differences in chromatographic mobility and to a wide range of biological activities. Gangliosides that accumulate in atherosclerotic lesions are characterized by aberrations in carbohydrate structure, with an increase in G_M3 and G_D3 (Figure 2, lane 1) compared with control normal tissue (lane 3). Atherosclerotic G_M3 migrates as a doublet with a prominent lower band in atherosclerotic aortic tissue (lane 1) and in tissue from an uninvolved area of the same aorta (lane 2) but not in tissue from control normal tissue (lane 3). The most likely explanation for this is a difference in the lipid (ceramide) composition of G_M3 from atherosclerotic tissue. The activity of these molecules might also be different owing to differences in lipid structure. We examined this possibility by comparing the effects of G_M3 and G_D3 isolated from AAGs versus the same carbohydrate structures isolated from NAGs. Figure 6 demonstrates that G_M3 from AAGs has a greater effect compared with G_M3 from NAGs (solid bars). Similarly, G_D3 from AAGs increased platelet adhesion to a greater degree compared with G_D3 from NAGs (Figure 6, hatched bars). Importantly, G_M3 and G_D3, irrespective of the source of the ganglioside molecule (AAGs or NAGs), both resulted in an increase in platelet adhesion to collagen compared with buffer controls (in the absence of gangliosides). These observations imply that both the carbohydrate portion and the ceramide moiety of the ganglioside molecule play a role in regulating the adhesive activity of the collagen-binding integrin ₂ß₁.

Gangliosides interact with a number of cell surface receptors, including integrins.³¹ We previously reported that circulating lipoprotein-associated gangliosides interact with platelets and enhance collagen-mediated activation.¹² Preincubation of platelets with circulating tumor-derived gangliosides resulted in more platelet aggregation and greater ATP release than in controls without gangliosides. Tumor gangliosides also enhance platelet adhesion to extracellular matrix collagen, the initial step in collagen-mediated platelet activation.³² Our prior published results combined with the data of others^{4 5 26} led us to speculate that accumulated gangliosides in atherosclerotic aortas might enhance platelet adhesion to the exposed extracellular matrix collagen in atherosclerotic blood vessels. In the present study, immobilized type I collagen fibers, similar to those found in the extracellular matrix of blood vessels, were used as the substrate for platelet adhesion to test this hypothesis. An increase of >2 orders of magnitude in platelet adhesion to collagen was observed after preincubation of platelets with AAGs compared with NAGs. Interestingly, similar enhancing effects were observed after preincubation of platelets with UAGs (Figure 3). The results of experiments performed with gangliosides isolated from diseased aortas, irrespective of whether the tissue used to isolate the gangliosides was grossly involved with plaque or not, demonstrated a significant enhancement in adhesion compared with experiments performed with NAGs or with buffer as a control. When these experiments were performed with the most prominent ganglioside structures identified in atherosclerotic tissue, G_M3 and G_D3, similar results were obtained, supporting the hypothesis that atherosclerotic gangliosides enhance platelet adhesion. This effect on platelet adhesion (Figure 4) was observed even at very low concentrations of ganglioside (eg, 0.5 µmol/L), which are within the physiological range observed in normal human serum.⁷

A large number of different platelet proteins have been proposed as possible receptors for collagen.^{23 33 34} The study of Coller et al²¹ suggested that the integrin collagen receptor ₂ß₁ (also referred to as very late antigen-2, VLA-2, or glycoprotein Ia/IIa) is the predominant receptor mediating platelet-collagen interactions in the absence of plasma. Because gangliosides are known to interact with a number of different integrins, including ₂ß₁, ₅ß₁, and _vß₃,^{31 35 36} we examined the possibility that AAGs might exert their enhancing influence on platelets through an effect on the integrin collagen receptor ₂ß₁. In these experiments, an anti-₂ monoclonal antibody, F-17, was used to block this receptor. In the presence of the ₂-blocking antibody, AAG-enhanced platelet adhesion was reduced to control levels (Figure 5); similar results were obtained for the major individual gangliosides G_M3 and G_D3 purified from AAGs (Figure 6). These findings suggest that atherosclerotic gangliosides enhance platelet adhesion to collagen by promoting the collagen-₂ß₁ interaction.

In summary, our results demonstrate that gangliosides accumulate in atherosclerotic aortic lesions and enhance platelet adhesion to collagen, the major component of the damaged vessel wall. Furthermore, we speculate that these accumulated atherosclerotic gangliosides play a role in plaque vulnerability by promoting thrombus formation and growth of the plaque after disruption or erosion. The fact that NAGs do not enhance platelet adhesion (Figure 4) whereas the individual gangliosides (G_M3 and G_D3) isolated from NAGs do increase platelet adhesion (Figure 6) suggests that it is the presence of increased amounts of specific molecules (ie, G_M3 and G_D3) that are responsible for the activity of the atherosclerotic gangliosides. Therefore, accumulated atherosclerotic gangliosides are potent stimulants of ₂ß₁-dependent platelet adhesion to collagen and point to a potential therapeutic target-ganglioside-modified ₂ß₁-dependent platelet-collagen interaction. This hypothesis is under investigation.

	Acknowledgments

 
This study was supported by American Heart Association Grant-in-Aid No. 96012790 to L.A.V. The authors wish to thank Marie Jones for her secretarial assistance and Ursula Balthazar-Stablein and Bharathi Nallapareddy, MBBS, for their helpful comments and editing of the manuscript.

Received March 10, 1998; accepted July 30, 1998.

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